The 1,5-anhydroglucitol (hereinafter "1,5-AG") is present in body fluids (or humors) such as serum, blood plasma, urine and so forth of humans, and its quantity in the body fluids greatly varies when one suffers from a certain disease, in particular, diabetes. Accordingly, in recent years, it is being an important diagnostic item in clinical diagnosis as a useful diagnostic marker.
As a method of quantitative assay for this 1,5-AG, a method is prevalent in which pyranose oxidase or L-sorbose oxidase is allowed to act on 1,5-AG and the hydrogen peroxide formed is subjected to calorimetric quantitative assay using a color-forming system using peroxidase (see Japanese Patent Publication No. 5-41238). In recent years, this method is applied to general-purpose automatic analyzers. Since, however, these enzymes have a low substrate specificity and may also strongly react with saccharides such as glucose, it has been necessary to beforehand completely remove or eliminate the saccharides in specimens.
Japanese Patent Publication No. 3-24200 also discloses a method in which a 1,5-AG oxidizing enzyme having a high specificity to the 1,5-AG, produced from the genus Pseudomonas sp. NK-85001, is used to quantitatively assay the amount of consumption of oxygen after its reaction with 1,5-AG or the reaction products such as a reduction product of an electron acceptor. However, the method of measuring the amount of consumption of oxygen is not suitable for treating many specimens continuously. The method of measuring the reduction product of an electron acceptor is also not suitable for practical application because the method can not be said to be satisfactory taking account of the reduction product itself which is reversible and unstable and in view of sensitivity, when ferricyanic compounds and dichlorophenolindophenol as disclosed in that publication are used as electron acceptors.
In an instance where the quantitative assay is made using an enzyme such as 1,5-AG dehydrogenase requiring NAD (nicotinamide adenine dinucleotide) as a coenzyme (see Japanese Patent Application Laid-open No. 2-268679), such an enzyme may be affected by enzymes requiring the NAD as a coenzyme, e.g., lactate dehydrogenase and the like, contained in a biological sample, and hence this method has a problem on accuracy.
It is also known that glucoside 3-dehydrogenase EC 1.1.99.13! confirmed to be present in the soluble fraction of the genus Agrobacterium (The Journal of Biological Chemistry, Vol.242, No.16, pp.3665-3672, 1967) or the membrane fraction of the genus Flavobacterium acts on the 1,5-AG (The Journal of Biochemistry, Vol.100, No.4, pp.1049-1055, 1986). Since, however, these enzymes also have a low substrate specificity, it is necessary to beforehand completely remove or eliminate the saccharides in specimens. Also, they require to use ferricyanic compounds or dichlorophenol indophenol as the electron acceptor, and have problems on safety and sensitivity.
Meanwhile, as the clinical significance of 1,5-AG, in diabetics the 1,5-AG concentration in serum or blood plasma specifically becomes as low as several .mu.g/ml. On the other hand, with regard to glucose concentration, it is about 100 mg/dl in the case of normal persons, whereas it may reach 1,000 mg/dl in the case of diabetics. Thus, the difference in concentration between 1,5-AG and glucose reaches as much as thousands of times. No enzymes have ever been discovered which enable direct quantitative assay for 1,5-AG and are perfectly specific to it in specimens in which glucose is present together in such a high concentration.
Then, the glucose must be completely removed or eliminated when the quantitative assay is made using the pyranose oxidase or L-sorbose oxidase, having a poor specificity to 1,5-AG and rather strongly reacting with glucose. For this purpose, various methods are proposed. For example, Japanese Patent Publication No. 5-41238 discloses a method in which a pre-treatment operation making use of an ion-exchanged resin is used in combination in order to remove saccharides such as glucose. As a method of eliminating glucose, Japanese Patent Application Laid-open No. 1-320998, Japanese Patent Publication No. 7-71514, Japanese Patent Applications Laid-open No. 6-237795, No. 3-27299, No. 6-245796, etc. disclose a method in which a glucose 6-position phosphorylated enzyme (glucokinase or hexokinase) is used and, also in order to bring this reaction into completion, the equilibrium of the phosphorylation of glucose is intended to be completely directed to glucose 6-phosphate (a coupling system employing several kinds of enzymes and substrates in combination). This method also intends to lessen the inhibitory action of ATP (adenosine 5'-triphosphate) on pyranose oxidase. Another method is also proposed as disclosed in Japanese Patent Publications No. 7-102154 and No. 7-108236, in which pH conditions for allowing pyranose oxidase to act on 1,5-AG are specified or pyranose oxidase with a different origin is used so that the ATP can have no inhibitory action.
However, of the above methods, the method employing an ion-exchanged resin requires so troublesome operations that it is not suitable for treating many specimens. Also, in the method employing a glucose 6-position phosphorylated enzyme and also a coupling system using several kinds of enzymes and substrates in combination, the reaction system is so complicated that the stability of the enzyme, substrate and so forth to be used must be taken into account. The method employing the glucose 6-position phosphorylated enzyme alone also have many restrictions such that the ATP must be used in great excess in order to completely convert and eliminate the glucose and the origin of pyranose oxidase and reaction conditions are limited. Thus, the method can not be so much general-purpose.